Lafora disease (LD) is a catastrophic epilepsy with onset in teenage years in otherwise normal adolescents. Its causes are known, its pathogenesis in advanced understanding, in major part through the research of members of this proposed program project. The disease genes, EPM2A and EPM2B, encode the laforin glycogen phosphatase and the malin ubiquitin E3 ligase, which regulate glycogen synthesis, in particular glycogen structure. Mutations of either gene lead to formation of malstructured glycogen (polyglucosan), which precipitates, aggregates, and accumulates into Lafora bodies (LB). These overtake neuronal somatodendritic cytoplasm and initiate a progressive soon-intractable epilepsy that aggressively escalates, and leads to death after 10 years of essentially constant seizing. Our groups have shown in the LD mouse models that reducing glycogen synthesis by 50-100% through knockouts of the glycogen synthase (GS) gene or the GS activator PTG gene are safe, and fully rescue murine LD. Here, we propose to translate these findings from gene knockout experiments to therapeutic interventions, in mouse, as a step towards human therapy. In Aim 1, we target the GS and Ptg genes at the DNA level through CRISPR/Cas9 genome editing, in collaboration with F Zhang, co- discoverer of the CRISPR system. We deliver CRISPR/Cas9 using the AAV9 virus, in collaboration with B Kaspar, an AAV9 field leader. We utilize a new Cas9 enzyme recently developed in the Zhang lab with a size that for the first time allows packaging in AAV9. In Aim 2, we target the GS and Ptg genes at the mRNA level using antisense oligonucleotides with unique brain-specific chemistries. Here, we already preliminarily show rescue. In Aim 3 we target glycogen itself. We utilize an innovative chain-termination approach, which introduces glucose derivatives into the growing chains of polyglucosans to block extension, and thus prevent their formation, and prevent LB and LD. Finally in Aim 4 we utilize the ketogenic diet, under which the brain utilizes ketones for energy in lieu of glucose and determine whether this can prevent LB formation. The aims of this and our other projects of this CWOW are a comprehensive intervention on the pathogenic pathway in LD. We expect that one, though more likely a combination, of these aims will succeed in our murine models and later translate to eliminating LD from the roster of intractable epilepsies.